Structure-Guided Interface Engineering of Formate Oxidase to Suppress Aggregation and Enhance Structural Stability

Formate oxidase (FOx) is an attractive biocatalyst for agricultural and food applications but suffers from severe activity loss at the high enzyme concentrations required for biosensors and in situ H₂O₂ generation. In this work, we combined biophysical assays, kinetic analysis, and molecular dynamics simulations to clarify the deactivation mechanism and guide interface engineering. FOx remains thermally stable at 25 °C, and the specific activity of the soluble fraction stays nearly constant, so the decline in total activity mainly arises from concentration-dependent aggregation, which removes enzyme from solution. Guided by this mechanism, we mapped aggregation-prone surface regions and used Rosetta to design 11 interface variants. Six variants reduce aggregation at 1 mg/mL, but most compromise activity; A378L is the only variant that reduces aggregation while maintaining decent catalytic efficiency by weakening AC-dimer packing without perturbing the active site. This interface-engineering framework improves FOx stability and is transferable to other multimeric enzymes.